Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.

Danish Cancer Society Research Center DK 2100 Copenhagen Denmark

Genomic Instability Group Spanish National Cancer Research Centre Madrid 28029 Spain

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University Olomouc Czech Republic

Pathology Department Complejo Hospitalario Universitario Insular Las Palmas Gran Canaria Spain

Science for Life Laboratory Division of Genome Biology Department of Medical Biochemistry and Biophysics Karolinska Institutet S 171 21 Stockholm Sweden

Zobrazit více v PubMed

Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, et al. Drug repurposing: progress, challenges and recommendations. Nat Rev Drug Discov. 2018;18:41–58. doi: 10.1038/nrd.2018.168. PubMed DOI

Skrott Z, Mistrik M, Andersen KK, Friis S, Majera D, Gursky J, et al. Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4. Nature. 2017;552:194–9. doi: 10.1038/nature25016. PubMed DOI PMC

Majera D, Skrott Z, Chroma K, Merchut-Maya JM, Mistrik M, Bartek J. Targeting the NPL4 adaptor of p97/VCP segregase by disulfiram as an emerging cancer vulnerability evokes replication stress and DNA damage while silencing the ATR pathway. Cells. 2020;9:469. doi: 10.3390/cells9020469. PubMed DOI PMC

Krastev DB, Li S, Sun Y, Wicks AJ, Hoslett G, Weekes D, et al. The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin. Nat Cell Biol. 2022;24:62–73. doi: 10.1038/s41556-021-00807-6. PubMed DOI PMC

Direito I, Monteiro L, Melo T, Figueira D, Lobo J, Enes V, et al. Protein aggregation patterns inform about breast cancer response to antiestrogens and reveal the rna ligase rtcb as mediator of acquired tamoxifen resistance. Cancers. 2021;13:3195. doi: 10.3390/cancers13133195. PubMed DOI PMC

Xu J, Reumers J, Couceiro JR, De Smet F, Gallardo R, Rudyak S, et al. Gain of function of mutant p53 by coaggregation with multiple tumor suppressors. Nat Chem Biol. 2011;7:285–95. doi: 10.1038/nchembio.546. PubMed DOI

Hurwitz B, Guzzi N, Gola A, Fiore VF, Sendoe A, Nikolova M, et al. The integrated stress response remodels the microtubule-organizing center to clear unfolded proteins following proteotoxic stress. Elife. 2022;11:77780. doi: 10.7554/eLife.77780. PubMed DOI PMC

Costa-Mattioli M, Walter P. The integrated stress response: from mechanism to disease. Science. 2020;368:1–11.. doi: 10.1126/science.aat5314. PubMed DOI PMC

Tiu GC, Kerr CH, Forester CM, Krishnarao PS, Rosenblatt HD, Raj N, et al. A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies. Dev Cell. 2021;56:2089–2102.e11. doi: 10.1016/j.devcel.2021.06.013. PubMed DOI PMC

Kasteri J, Das D, Zhong X, Persaud L, Francis A, Muharam H, et al. Translation control by p53. Cancers. 2018;10:133. doi: 10.3390/cancers10050133. PubMed DOI PMC

Guan BJ, Krokowski D, Majumder M, Schmotzer CL, Kimball SR, Merrick WC, et al. Translational control during endoplasmic reticulum stress beyond phosphorylation of the translation initiation factor eif2. J Biol Chem. 2014;289:12593–611. doi: 10.1074/jbc.M113.543215. PubMed DOI PMC

Heyer EE, Moore MJ. Redefining the translational status of 80S monosomes. Cell. 2016;164:757–69. doi: 10.1016/j.cell.2016.01.003. PubMed DOI

Pakos‐Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM. The integrated stress response. EMBO Rep. 2016;17:1374–95. doi: 10.15252/embr.201642195. PubMed DOI PMC

Gnanasundram SV, Fåhraeus R. Translation stress regulates ribosome synthesis and cell proliferation. Int J Mol Sci. 2018;19:3757. doi: 10.3390/ijms19123757. PubMed DOI PMC

Yang K, Yang J, Yi J. Nucleolar Stress: hallmarks, sensing mechanism and diseases. Cell Stress. 2018;2:125–40. doi: 10.15698/cst2018.06.139. PubMed DOI PMC

Kanellis DC, Espinoza JA, Zisi A, Sakkas E, Bartkova J, Katsori AM, et al. The exon-junction complex helicase eIF4A3 controls cell fate via coordinated regulation of ribosome biogenesis and translational output. Sci Adv. 2021;7:1–19.. doi: 10.1126/sciadv.abf7561. PubMed DOI PMC

Lindström MS, Bartek J, Maya-Mendoza A. p53 at the crossroad of DNA replication and ribosome biogenesis stress pathways. Cell Death Differ. 2022;29:972–82. doi: 10.1038/s41418-022-00999-w. PubMed DOI PMC

Michael D, Oren M. The p53-Mdm2 module and the ubiquitin system. Semin Cancer Biol. 2003;13:49–58. doi: 10.1016/S1044-579X(02)00099-8. PubMed DOI

Bartek J, Falck J, Lukas J. CHK2 kinase-a busy messenger. Nat Rev Mol Cell Biol. 2001;2:877–86. doi: 10.1038/35103059. PubMed DOI

Liu Y, Tavana O, Gu W. p53 modifications: exquisite decorations of the powerful guardian. J Mol Cell Biol. 2019;11:564–77. doi: 10.1093/jmcb/mjz060. PubMed DOI PMC

Corsello SM, Nagari RT, Spangler RD, Rossen J, Kocak M, Bryan JG, et al. Discovering the anticancer potential of non-oncology drugs by systematic viability profiling. Nat Cancer. 2020;1:235–48. doi: 10.1038/s43018-019-0018-6. PubMed DOI PMC

Buchtova T, Skrott Z, Chroma K, Rehulka J, Dzubak P, Hajduch M, et al. Cannabidiol-induced activation of the metallothionein pathway impedes anticancer effects of disulfiram and its metabolite CuET. Mol Oncol. 2022;16:1541–54. doi: 10.1002/1878-0261.13114. PubMed DOI PMC

Guan BJ, van Hoef V, Jobava R, Elroy-Stein O, Valasek LS, Cargnello M, et al. A unique ISR program determines cellular responses to chronic stress. Mol Cell. 2017;68:885–900.e6. doi: 10.1016/j.molcel.2017.11.007. PubMed DOI PMC

Ferreira R, Schneekloth JS, Panov KI, Hannan KM, Hannan RD. Targeting the RNA polymerase I transcription for cancer therapy comes of age. Cells. 2020;9:226:249. doi: 10.3390/cells9020266. PubMed DOI PMC

Liao H, Gaur A, Mauvais C, Denicourt C. P53 induces a survival transcriptional response after nucleolar stress. Mol Biol Cell. 2021;32:1–11.. doi: 10.1091/mbc.E21-05-0251. PubMed DOI PMC

Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, et al. Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis. 2013;4:e838–e838. doi: 10.1038/cddis.2013.350. PubMed DOI PMC

Mauthe M, Orhon I, Rocchi C, Zhou X, Luhr M, Hijlkema KJ, et al. Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy. 2018;14:1435–55. doi: 10.1080/15548627.2018.1474314. PubMed DOI PMC

Espinoza JA, Zisi A, Kanellis DC, Carreras-Puigvert J, Henriksson M, Hühn D, et al. The antimalarial drug amodiaquine stabilizes p53 through ribosome biogenesis stress, independently of its autophagy-inhibitory activity. Cell Death Differ. 2020;27:773–89. doi: 10.1038/s41418-019-0387-5. PubMed DOI PMC

Yang-Hartwich Y, Soteras MG, Lin ZP, Holmberg J, Sumi N, Craveiro V, et al. p53 protein aggregation promotes platinum resistance in ovarian cancer. Oncogene. 2015;34:3605–16. doi: 10.1038/onc.2014.296. PubMed DOI

Skrott Z, Majera D, Gursky J, Buchtova T, Hajduch M, Mistrik M, et al. Disulfiram’s anti-cancer activity reflects targeting NPL4, not inhibition of aldehyde dehydrogenase. Oncogene. 2019;38:6711–22. doi: 10.1038/s41388-019-0915-2. PubMed DOI

Ianevski A, Giri AK, Aittokallio T. SynergyFinder 2.0: visual analytics of multi-drug combination synergies. Nucleic Acids Res. 2020;48:W488–W493.. doi: 10.1093/nar/gkaa216. PubMed DOI PMC

Ewels PA, Peltzer A, Fillinger S, Alneberg J, Patel H, Wilm A et al. nf-core: community curated bioinformatics pipelines. Nat Biotechnol. 2020;38:276–278. PubMed

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:1–21.. doi: 10.1186/s13059-014-0550-8. PubMed DOI PMC

Raudvere U, Kolberg L, Kuzmin I, Arak T, Adler P, Peterson H, et al. G:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update) Nucleic Acids Res. 2019;47:W191–W198.. doi: 10.1093/nar/gkz369. PubMed DOI PMC

Walter W, Sánchez-Cabo F, Ricote M. GOplot: an R package for visually combining expression data with functional analysis. Bioinformatics. 2015;31:2912–4. doi: 10.1093/bioinformatics/btv300. PubMed DOI

Nueda MJ, Tarazona S, Conesa A. Next maSigPro: updating maSigPro bioconductor package for RNA-seq time series. Bioinformatics. 2014;30:2598–602. doi: 10.1093/bioinformatics/btu333. PubMed DOI PMC

Gandin V, Sikström K, Alain T, Morita M, McLaughlan S, Larsson O, et al. Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale. JoVE. 2014;17:e51455–e51464.. PubMed PMC

Bartkova J, Horejsí Z, Koed K, Krämer A, Tort F, Zieger K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature. 2005;434:864–70. doi: 10.1038/nature03482. PubMed DOI

Paes Dias M, Tripathi V, van der Heijden I, Cong K, Manolika EM, Bhin J, et al. Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps. Mol Cell. 2021;81:4692–4708.e9. doi: 10.1016/j.molcel.2021.09.005. PubMed DOI PMC

Kosar M, Giannattasio M, Piccini D, Maya-Mendoza A, García-Benítez F, Bartkova J, et al. The human nucleoporin Tpr protects cells from RNA-mediated replication stress. Nat Commun. 2021;12:1–18.. doi: 10.1038/s41467-021-24224-3. PubMed DOI PMC

Pudelko L, Edwards S, Balan M, Nyqvist D, Al-Saadi J, Dittmer J, et al. An orthotopic glioblastoma animal model suitable for high-throughput screenings. Neuro Oncol. 2018;20:1475–84. doi: 10.1093/neuonc/noy071. PubMed DOI PMC

Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinforma. 2017;18:1–26.. doi: 10.1186/s12859-017-1934-z. PubMed DOI PMC

Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD Guidelines for the Testing of Chemicals, Section 2: Effects on Biotic Systems. OECD iLibrary. https://www.oecd-ilibrary.org/environment/test-no-236-fish-embryo-acute-toxicity-fet-test_9789264203709-en. Accessed 27 Mar 2023.

Kwak SG, Kim JH. Central limit theorem: the cornerstone of modern statistics. Korean J Anesthesiol. 2017;70:144–56. doi: 10.4097/kjae.2017.70.2.144. PubMed DOI PMC